Serotype-specific identification of enterovirus 71 by PCR☆
Introduction
The two leading agents responsible for large outbreaks of hand, foot, and mouth disease (HFMD), a common, usually benign, rash illness in children, are the enteroviruses coxsackievirus A16 (CA16) and enterovirus 71 (EV71). CA16 and EV71 are closely related genetically, but EV71 is also associated with severe neurologic disease, such as encephalitis, meningitis, cranial nerve palsies, Guillain–Barre syndrome, and poliomyelitis-like syndrome much more frequently than is CA16 (reviewed in Chumakov et al., 1979, Gilbert et al., 1988, Alexander et al., 1994, Landry et al., 1995). Two recent EV71 disease outbreaks in Asia (Malaysia, 1997, and Taiwan, 1998) have involved thousands of HFMD cases, some of which were associated with severe neurologic disease and rapid death (WHO, 1997, CDC, 1998, Chang et al., 1998, Lum et al., 1998).
Because of increased public health concerns associated with EV71 and its potential for greater neurovirulence, there is a need for a diagnostic method that can rapidly discriminate between EV71 and CA16 during a large HFMD outbreak. Since the symptoms of EV71- and CA16-associated HFMD are the same, the etiologic diagnosis depends on virus isolation and serotyping. Standard serotyping involves neutralization tests with monospecific antiserum, reagents that are quite limited in their availability. Furthermore, antigenic typing is often hampered by nonneutralizable virus due to aggregation (Blomberg et al., 1974, Schmidt et al., 1974, Nagy et al., 1982). A second typing method has used immunofluorescence techniques utilizing commercially available monoclonal antibodies. End-labeled nucleic acid probes in the VP2 region have also been used in diagnostic tests for EV71 and CA16 (Kitamura et al., 1997).
We report here the development of an EV71-specific RT-PCR assay, using primers in and near the VP1 region. We have developed and evaluated two primer pairs, one that is designed for a rapid-screening, EV71-specific RT-PCR assay and another that is designed for amplification and partial sequencing of VP1 in support of molecular epidemiology studies.
Section snippets
Viruses
The 125 EV71 strains examined in this study include 114 strains previously characterized (Brown et al., 1999, manuscript submitted) and the following additional isolates, with year and state or country of isolation: OK97-2354; CA87-3105; CA88-3104; CA90-3103; MD87-9256; and TAI98-2731; TAI98-2732; TAI98-2733; TAI98-2734; TAI98-2735 and TAI98-2785. The 125 EV71 strains were isolated between 1970 and 1998 at the Centers for Disease Control and Prevention, Atlanta, Georgia, from 25 different
Results
Since enterovirus serotypes are defined by neutralization using immune sera directed against the capsid proteins, nucleotide and amino acid sequences of the capsid region correlate with serotype (Oberste et al., 1999a). In particular, the VP1 -coding region has been shown to specifically correlate with serotype (Oberste et al., 1999a), and this region has been successfully targeted for the development of molecular typing reagents (Kilpatrick et al., 1996, Kilpatrick et al., 1998, Oberste et
Discussion
Awareness of EV71 as an agent of severe neurological disease has increased in the wake of the elimination of poliovirus from the Americas (da Silva et al., 1996) and following fatalities associated with large HFMD outbreaks in Asia (WHO, 1997, CDC, 1998, Chang et al., 1998, Lum et al., 1998). Rapid identification of enteroviruses isolated from paralytic cases in areas no longer endemic for wild poliovirus circulation is essential to the final success of the Global Poliomyelitis Eradication
Acknowledgements
The authors would like to acknowledge laboratories that have provided viruses for screening for EV71. We thank Candra Street for technical assistance.
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This information was presented, in part, at the annual meeting of the American Society for Virology, July 19, 1997, in Bozeman, Montana, USA (poster P14-3).